Multivalent vaccine composition having increased stability of capsular polysaccharide

ABSTRACT

A vaccine composition comprising two valences is provided: (i) a first valence which is adjuvant-enhanced with aluminum hydroxide and (ii) a second valence which contains a polysaccharide of bacterial capsule comprising one or more o-acetyl groups and which is not adsorbed with aluminum oxide due to the presence of a protecting compound which may be a phosphate, a citrate or a carbonate and which prevents the adsorption. The first valence can be any vaccine valence. In one particular embodiment, the vaccine composition contains (i) Hepatitis A valence, adsorbed on aluminum hydroxide and (ii) the typhoid fever valence formed by the polysaccharide Vi of the  Salmonella typhi  capsule.

The present invention relates in particular to a stabilized bivalenthepatitis A/typhoid fever (HA-Vi) immunization composition in which theVi valence conserves its immunogenic power for at least 24 monthsapproximately.

Hepatitis A and typhoid fever are two diseases for which a vaccinealready exists. They are both found in regions of the world where thehygiene conditions are far from optimal. Since the respective infectiousagents have in common the same route of transmission (oral-fecal route)and since the areas endemic for these diseases greatly overlap, itappears to be advantageous to combine the two valences in the sameproduct. In particular, it will easily be understood that, in the rangeof vaccines for travelers, an HA-Vi combination is more attractive thanthe two monovalent HA and Vi vaccines which have to be administeredseparately.

Studies have already been carried out in order to verify-that the HA andVi valences are compatible in particular in terms of inocuity, ofimmunogenic power and of stability. These studies use HA-Vi combinationsprepared from the monovalent vaccines which already exist on the market;there are essentially two of these studies; firstly, the studies carriedout by combining the Havrix™ (HA) and (Vi) monovalent vaccines producedby SmithKline Beecham Biologicals (Rixensart, Belgium) and, secondly,the studies carried out by combining the Avaxim™ (HA) and Typhim Vi™(Vi) monovalent vaccines produced by Aventis Pasteur (Lyons, France).

In the two series of studies, the monovalent HA vaccine consists ofinactivated hepatitis A virus adsorbed onto aluminum hydroxide.Similarly, the monovalent typhoid fever vaccine consists of Salmonellatyphi capsular polysaccharide which remains nonadjuvanted. Finally, thebivalent combinations are produced in an identical way, by simply mixingthe corresponding monovalent vaccines (these monovalent vaccines aremarketed in liquid form). Thus, a dose of Avaxim™ and a dose of TyphimVi™ are mixed together to give a dose of the bivalent HA-Vi combination.

The two series of studies were carried out with combinations made upless than twenty months before giving the injections in the context ofclinical trials. These studies showed that the bivalent combinationswere equivalent to the corresponding monovalent vaccines, in particularin terms of immunogenic power. Thus, the bivalent combination ofSmithKline Beecham Biologicals satisfied the requirements of the Britishauthorities and has already received a marketing authorization in thiscountry. However, this authorization has the restriction of an expirydate set at 12 months after manufacture.

In the field of vaccines, an expiry date of 12 months cannot beconsidered to be sufficient given, in particular, the time required forcontrolling the batches before distribution. An expiry date set at 24;or even better, 36 months, greatly facilitates the marketing of thebatches.

Now, in the case of the HA-Vi combinations, the applicant has noticedthat, beyond 16-18 months past the date of manufacture, the Vi valencegradually loses its immunogenic power, and has put forward a hypothesisregarding the cause of this instability. Specifically, the O-acetylgroups of the Vi polysaccharide, which are characteristic of theimmunogenicity of the Vi, hydrolyze over time, especially under alkalineconditions. This hydrolysis is considered to be responsible for thedecrease in immunogenic power of the Vi polysaccharide and is thought tobe due to the adsorption of the Vi component onto the aluminum hydroxidewhich is present in the bivalent combination as an adjuvant of the HAvalence. The Vi valence immediately adsorbs onto the aluminum gel assoon as the monovalent vaccines are mixed with one another. Thisadsorption causes the Vi polysaccharide to be maintained in an alkalineenvironment. Specifically, since the aluminum hydroxide is positivelycharged, it attracts the OH— ions of the medium, which brings about anincrease in the pH in the microenvironment of the aluminum gel in whichthe Vi is located subsequent to its adsorption. As for the hydrolysis ofthe O-acetyls, this is a very slow phenomenon, the effects of which arereally noticeable after 16-18 months approximately.

Not only has the applicant demonstrated the problem of the instabilityof the Vi valence over time, but it provides a solution which consistsin adding to the bivalent combination a compound which prevents the Vivalence adsorbing onto the aluminum gel, while at the same timemaintaining the HA valence in an adjuvanted form. Advantageously, thiscompound may be an anion, such as a phosphate or citrate ion.

In its most general teaching, the invention therefore relates to animmunization composition comprising at least two valences; (i) a firstvalence which is adjuvanted with aluminum hydroxide and (ii) a secondvalence which contains a bacterial capsular polysaccharide comprisingone or more O-acetyl groups and which is not adsorbed onto the aluminumhydroxide by virtue of which the first valence is adjuvanted, due to thepresence of an additional compound which prevents the adsorption of thesecond valence onto the aluminum hydroxide, without disturbing theadsorption of the first valence.

According to an advantageous embodiment, the adsorption of the secondvalence may be prevented by the presence of a protective anioniccompound, on condition that it has all the safety guarantees requiredfor use for immunization purposes. This protective compound may be aphosphate, a citrate or a carbonate. It is also possible to use acombination of various anions, for example a combination of phosphateand citrate ions. By way of indication, it is specified that phosphateions may in particular be provided by a solution containingmonopotassium phosphate, disodium phosphate and sodium chloride.

The use of a protective compound in particular makes it possible tostabilize the antigenic activity of the second valence over a longperiod (24 months or more), preferably during conservation at normalstorage temperature or higher (e.g. 37° C.). The stability of theimmunogenic activity can be estimated, using various techniques, bymeasuring this activity at the time the composition is produced and thenperforming identical measurements over time, or at least 24 months afterthe date of production, and comparing the results obtained. When thedetailed figures are not established as being statistically differentfrom one another, then it should be considered that the immunogenicactivity is stable. The immunogenic activity of an antigen can inparticular be assayed by the ELISA technique, used commonly in thevaccines field.

The first valence may be any vaccine valence without restriction of typeor of structure, on condition of course that it needs to be adjuvanted.Mention is in particular made of the hepatitis A (HepA or HA) valence,the hepatitis B (HepB) valence and the pneumococcus valence. This firstvalence may consist of an inactivated virus, such as the inactivated HAvirus or the inactivated polio virus; an attenuated virus; a viral orbacterial subunit antigen, such as the hepatitis B virus surfaceantigen, or the diphtheria or tetanus toxoid.

The second valence consists, by definition, of a polysaccharide, whichmay or may not be conjugated, which contains one or more O-acetyl groupswithin its repeat unit. The Salmonella typhi capsule of polysaccharide(also called Vi polysaccharide) and the Neisseria meningitidis group Acapsule of polysaccharide satisfy this definition. In these cases,reference is therefore made to Vi or typhoid fever valence and tomeningo A valence.

The term “bacterial capsular polysaccharide” is intended to mean apolysaccharide consisting of the chain of the repeat unit characteristicof a capsular polysaccharide, whatever its size and independently of anysupplementary modification. The repeat unit of a polysaccharide makingup the composition according to the invention necessarily comprises atleast one O-acetyl group.

For the purposes of the present invention, the polysaccharide may beobtained in purified form from the bacterium of origin according toentirely conventional techniques. Depending on needs, the polysaccharidemay be (i) fragmented or unfragmented and (ii) conjugated or notconjugated to a carrier polypeptide such as diphtheria or tetanustoxoid.

In a more specific context, a subject of the invention is animmunization composition comprising (i) the HA valence adjuvanted withaluminum hydroxide and (ii) the typhoid fever valence consisting of Vipolysaccharide; this being a composition in which the Vi valence is notadsorbed onto the aluminum hydroxide.

For the purposes of the present invention, the first valence may beadjuvanted either by precipitation with the aluminum hydroxide or byadsorption onto the aluminum hydroxide.

The aluminum hydroxide used to adjuvant the first valence may be purealuminum hydroxide (i.e. an aluminum compound comprising only A13+ ionsand hydroxide groups) or any aluminum known under this name, even if,from a chemical point of view, they do not consist exclusively ofaluminum hydroxide. Thus, they may also be mixed aluminum compounds,such as those denoted under the name aluminum hydroxyphosphate orhydroxysulfate. In general, they may be any aluminum compoundcomprising, in particular, hydroxide groups. By way of illustration,mention may be made of the aluminum hydroxide Alhydrogel™ marketed bythe company Superfos Biosector.

The phosphate, citrate, or carbonate ions (protective compound) must beadded in sufficient amount to prevent the adsorption of the secondvalence while at the same time maintaining the first valence in anadjuvanted form. This amount depends on various factors, among which arethe amount and nature of the first valence, its adjuvanting method, theamount and nature of the aluminum hydroxide and the amount of the secondvalence. Those skilled in the art in the field of vaccines are entirelycapable of taking these constraints into account in order to determinethe suitable amount of the compound preventing the adsorption of thesecond valence, once the other factors have been established, such thatthe aluminum hydroxide is saturated with the phosphate ions while at thesame time maintaining the first valence in the adjuvanted state.

However, it is specified that the commercial vaccines conventionallycontain from 0.6 to 1.5 mg of aluminum/ml. In the commercial HepAvaccines, the alumina gel, present at the conventional doses (expressedas amount of aluminum), is in great excess compared to the HepA antigensince the adsorption sites of the alumina gel are far from beingsaturated. Thus, in practice, only the amount of aluminum appears to bedeterminant in establishing the amount of protective compound which mustbe present.

For a composition according to the invention containing 0.3 mg ofaluminum in the form of aluminum hydroxide and in a volume of 0.5 ml,approximately 20 mM of phosphate ions should be added. If the dose ofaluminum is doubled in this same volume, twice as many phosphate ionsshould be added, i.e. 40 mM. However, for a composition according to theinvention containing 0.6 mg of aluminum in a volume of 1 ml, 20 mM ofphosphate ions is sufficient.

By way of illustration, it is indicated that a bivalent vaccineaccording to the invention may contain, in a volume of 0.5 ml, (i) 160antigenic units or 1440 ELISA units of inactivated hepatitis A virus,adsorbed onto (ii) aluminum hydroxide containing 0.3 mg of aluminum;(iii) 0.025 mg of Vi polysaccharide; and (iv) 20 mM of phosphate ions.

The antigenic units and the ELISA units mentioned above are,respectively, units established using reference ELISA assays specific tothe companies Aventis Pasteur and SmithKline Beecham Biologicals (vanHoecke et al., J. Travel. Med. (1998) 5: 116 and André et al., in Prog.Med. Virol., Melnick J L Ed, Basle, Karger (1990) 37:72). It cannot beotherwise since there is no standardized reference for the HepA vaccine.

A composition according to the invention is advantageously in a liquidform, and an immunization dose is advantageously formulated in a volumeof between 0.5 and 1 ml, inclusive.

A composition according to the invention may be prepared by:

-   (i) adding phosphate, citrate or carbonate ions to a preparation    containing a valence other than the typhoid fever valence,    adjuvanted with an aluminum hydroxide; and-   (ii) mixing the preparation obtained in point (i) with a preparation    containing the typhoid fever valence.

EXAMPLE Preparation of an HA Vi Composition According to the Invention

A—Preparation of the Adsorbed HA Component

99 ml of a batch of inactivated HA virus, the antigenic titer of whichis 885 ELISA U/ml, is mixed with 5.94 ml of 2-phenoxyethanol at 25%.Homogenization is carried out for 15 min and the preparation is thenfiltered over a Millipak 40 MPGL 04SH2 filter. After filtration, 95 mlof a preparation with a titer of 835 ELISA U/ml are obtained.

47.5 ml of an alumina gel containing 3.14 mg of aluminum/ml are added tothis preparation. The volume is made up with 48 ml of 40 mM PBS(Phosphate Buffer). The mixture is stirred overnight at 5° C. (18 hours)in order for the HA component to adsorb onto the alumina gel. The pH is7.28.

B—Preparation of a 10-Times Concentrated Solution of Vi Polysaccharide

A Vi polysaccharide powder is prepared according to the method ofGotschlich et al., Prog. Immunobiol. Standard (1972) 5:485. 25.1 ml ofdistilled water prepared for injection (pfi) are poured little by littleinto a 30 ml flask containing 16.64 mg of Vi (9.5 g % of residual water,i.e. 15.06 mg of dry weight), with continual stirring. The stirring isallowed to continue for 24 hours at 5° C. in order to obtain completedissolution of the polysaccharide. This preparation is filtered over a0.22 μm Millex GV SLGV 025 filter. The filter is rinsed with 5 ml of pfidistilled water. The final volume is therefore 30.1 ml.

C—Preparation of the Bivalent HAVi Vaccine

10.6 ml of a 94 mM phosphate solution, followed by 8.1 ml of thepreparation obtained in point B) (Vi), are added to 62 ml of thepreparation obtained in point A (HA). The characteristics of the vaccinethus obtained are as follows:

Final phosphate concentration: 20 mM

pH: 7.3

Osmolarity: 578 mosm/kg

This preparation, named preparation P2; is then divided up into 0.5 mldoses.

In parallel, two other bivalent compositions, P2′ and P2″, were alsoprepared, no longer containing 20 mM of phosphate, but 10 and 40 mM ofphosphate, respectively. To do this, 10.6 ml of a phosphate solution at17.6 mM or 246 mM, depending on the species, are added.

The behavior of the HA and Vi components in the P2; P2′ and P2″compositions was immediately studied by assaying these components, byELISA, in the compositions without further manipulation or aftercentrifugation. It was noted that, at 10 mM of phosphate, the HAremained adsorbed but Vi adsorbed onto the alumina gel, whereas, at 40mM of phosphate, the Vi no longer adsorbed but the HA partiallydesorbed. At 20 mM of phosphate, the desired conditions are satisfied:the HA remains adsorbed whereas the Vi does not adsorb.

Subsequently, the stability of the P2 doses was studied at 5° C.±3° C.for 30 months. The antigenic capacity of the Vi polysaccharide in theimmunization doses taken in their entirety, and in the supernatant aftercentrifugation (the component adsorbed onto the alumina gel sedimentswith the gel), was in particular evaluated by ELISA; this also makes itpossible to establish the percentage of Vi not adsorbed.

The indirect ELISA method is used. The Vi antigen to be assayed issandwiched between anti-Vi antibodies covering the bottom of a plate andmouse anti-Vi antibodies. A biotinylated anti-mouse-IgG antibody isadded, followed by the horseradish peroxidase-coupled biotinylatedstreptavidin complex. The reaction is revealed by adding the substrateorthophenylenediamine dihydrochloride (OPD). The degradation of the OPDcauses an orangy-brown coloration proportional to the amount of Viantigen. Its intensity is measured on a spectrophotometer.

100 μl of an anti-Salmonella typhi serum are distributed into 96-wellplates. This is left to incubate for 5 hours at 37° C. and then theplate is emptied and 3 washes in PBS (phosphate buffer) containing 0.05%Tween are carried out. The free sites are saturated by adding 200 μl ofa solution of powdered milk diluted in PBS. Incubation is performed for1 hr 30 minutes at 37° C. and then the plate. is emptied and 3 washesare performed. Doubling serial dilutions of a standard vaccine areprepared so as to obtain a standard range. The doses of vaccine to beassayed and also a reference dose (which makes it possible to verify thecalibration range) are suitably diluted; 100 μl of each dilution aredistributed into the cupules and left to incubate overnight at 37° C.The plate is emptied and washes are performed. 100 μl per cupule of ananti-Vi mouse serum, suitably diluted, are then added. This is left toincubate for 1 hr at 37° C. and then the plate is emptied and washes areperformed. Biotinylated anti-mouse immunoglobulins are then attached(100 μl per cupule of a suitable dilution). This is left to incubate for1 hour at 37° C. and then the plate is emptied and washes are performed.Peroxidase-coupled biotinylated streptavidin is then attached (100 μlper cupule of a suitable dilution). This is left to incubate for 1 hourat 37° C. and then the plate is emptied and washes are performed. Theplate is developed by adding 100 μl per cupule of a solution of OPD at 1mg/ml in citrate phosphate buffer, pH 5. This is left to incubate for 30min at ambient temperature in the dark before adding 100 μl of 2 Nsulfuric acid to the cupules. The plate is read on a spectrophotometerat 492 nm. The standard curve of adsorbance as a function ofconcentration is established. The titer of each of the dilutions assayedis calculated relative to the standard curve and is expressed in ng/ml.In order to obtain the mean titer for each immunization dose assayed,the mean of the titers obtained with all the dilutions is calculated.The titer is given a μg/dose.

The amount of Vi polysaccharide O-acetyls present in the supernatant,after certification, was also measured. The O-acetyls are titered with acolorimetric method using hydroxylamine (Hestrin S. J. Biol. Chim.(1949) 180: 249). Hydroxylamine in alkaline medium forms, with esters, ahydroxamic acid which, in the presence of ferric salt, gives a browncoloration, the intensity of which is measured on a spectrophotometer at540 nm.

In parallel, an identical study was carried out with a preparationtermed preparation P1; prepared in the same way as P2; with the onlydifference being that phosphate is not added at the time the bivalentvaccine is made up. In this case, the Vi component immediately adsorbsonto the alumina gel and, in order to assay it after centrifugation, asfor P2; it should be desorbed beforehand. This desorption is obtained bymodifying the pH and the ionic strength of the medium. Aftercentrifugation, the alumina gel is brought into contact with a 150 mMtrisodium citrate solution for 6 hours at 37° C. The mixture is thencentrifuged in order to collect the supernatant in which the Vicomponent is found.

The results are given in Table I below.

TABLE I 3 6 9 12 18 24 30 5° C. T0 months months months months monthsmonths months Vi in the Vi ELISA P2 23.6 25.5 21.7 21.3 18.5 23.7 24.326.5 whole (·g/dose) P1 24.4 23.3 22.2 19.8 17.9 20.5 18.6 19.3 vaccineVi in the Vi ELISA P2 24.3 26 23.1 21.8 17.9 25 21.9 25.8 supernatant(·g/dose) P1 21 16.9 16.5 15.4 12 14.7 21.8 15.7 after O-acetyls P20.127 0.148 0.116 0.117 0.130 0.134 0.095 0.143 centrifugation(·mol/dose) P1 0.081 0.056 0.055 0.055 0.061 0.057 0.090 0.054Polysaccharides P2 32.5 30.5 27.6 31 30.4 29.1 31.7 29.3 (·g/dose) P120.8 22 15.6 16.2 18.5 16.5 17 18.4 % desorption of the Vi in P1 86% 72%74% 78% 67% 72% 63% 81.6%

The stability of the P2 formulation was also studied at 25° C.±2° C. for6 months and at 37° C.±3° C. for 3 months. The results are given inTables II and III below.

TABLE II 1 3 6 25 ° C. T0 month months months Vi in the Vi ELISA P2 23.623.6 21.7 20.6 whole (μg/dose) P1 24.4 19.6 11.3 7.4 vaccine Vi in theVi ELISA P2 24.3 25.5 22.6 20.1 supernatant (μg/dose) P1 21 15 10.3 6.3after O-acetyls P2 0.127 0.130 0.103 0.137 centrifugation (μmol/dose) P10.081 0.053 0.041 0.029 Poly- P2 32.5 31.6 25.5 27.2 saccharides P1 20.817.3 10.3 6.3 (μg/dose) % desorption of the 86% 77% 91% 85% Vi in P1

TABLE III 37° C. T0 1 month 3 months Vi in the Vi ELISA P2 23.6 24.1 21whole (μg/dose) P1 24.4 12.7 5.6 vaccine Vi in the Vi ELISA P2 24.3 25.521.1 supernatant (μg/dose) P1 21 9.9 4.7 after O-acetyls P2 0.127 0.1430.104 centrifugation (μmol/dose) P1 0.081 0.050 0.026 Polysaccharides P232.5 36 22.5 (μg/dose) P1 20.8 15.4 9.9 % desorption of the Vi 86% 78%84% in P1

1. An immunization composition comprising (i) a first valence adjuvantedwith an aluminum compound comprising hydroxide groups, (ii) a secondvalence comprising a bacterial capsule polysaccharide comprising one ormore O-acetyl groups, and (iii) phosphate, citrate, and/or carbonateions in an amount sufficient to prevent adsorption of the second valenceonto the aluminum compound, wherein the first valence is hepatitis Avalence and the second valence is a typhoid fever Salmonella typhicapsular Vi polvsaccharide.
 2. The composition of claim 1 wherein thehepatitis A valence is inactivated hepatitis A virus.
 3. The compositionof claim 1 or 2 wherein the typhoid fever valence has a stable antigenictiter for at least 24 months.
 4. The composition of claim 1 or 2 whereinthe first valence is adjuvanted with an aluminum compound which isaluminum hydroxide or aluminum hydroxyphosphate.
 5. The composition ofclaim 1 or 2 wherein the first valence is adjuvanted by adsorption ontoan aluminum compound.
 6. A method for manufacturing the composition ofclaim 1 the method comprising: (a) adding phosphate, citrate, and/orcarbonate ions to a preparation comprising a first valence adjuvantedwith an aluminum compound comprising hydroxide groups; and (b) mixingthe preparation obtained in step (a) with a preparation comprising asecond valence comprising a bacterial capsular polysaccharide comprisingone or more O-acetyl groups, wherein the amount of phosphate, citrate orcarbonate ions added is sufficient to prevent adsorption of the secondvalence onto the aluminum compound, and wherein the first valence ishepatitis A valence and the second valence is typhoid fever Salmonellatyphi capsular Vi polvsaccharide.
 7. The method of claim 6 wherein thehepatitis A valence is inactivated hepatitis A virus.
 8. The method ofclaim 6 wherein the typhoid fever valence has a stable antigenic titerfor at least 24 months.
 9. The method of claim 6 wherein the firstvalence is adjuvanted with an aluminum compound which is aluminumhydroxide or aluminum hydroxyphosphate.
 10. The method of claim 6wherein the first valence is adjuvanted by adsorption onto an aluminumcompound.